Some implementations of interference microscopy imaging use digital holographic measurements of complex scattered fields to reconstruct three-dimensional refractive index maps of weakly scattering, semi-transparent objects, frequently encountered in biological investigations. Reconstruction occurs through application of the object scattering potential which assumes an isotropic refractive index throughout the object. Here, we demonstrate that this assumption can in some circumstances be invalid for biological imaging due to the presence of lipid-induced optical anisotropy. We show that the nanoscale organization of lipids in the observation of cellular endocytosis with polarized light induces a significant change in far-field scattering. We obtain this result by presenting a general solution to Maxwell's equations describing light scattering of core–shell particles near an isotropic substrate covered with an anisotropic thin film. This solution is based on an extension of the Bobbert–Vlieger solution for particle scattering near a substrate delivering an exact solution to the scattering problem in the near field as well as far field. By applying this solution to study light scattering by a lipid vesicle near a lipid bilayer, whereby the lipids are represented through a biaxial optical model, we conclude through ellipsometry concepts that effective amounts of lipid-induced optical anisotropy significantly alter far-field optical scattering in respect to an equivalent optical model that neglects the presence of optical anisotropy.Lay description:
After the demonstration of super-resolution fluorescence microscopy imaging leading to the award of the Nobel prize in chemistry in 2014, there has been a strong renewed interest in interference microscopy techniques that do not exhibit limitations of fluorescence staining and disruptive fluorescence photophysics phenomena including photobleaching and blinking. One of these interferometry based microscopy techniques known as Tomographic Phase Microscopy (TPM) has recently been explored for applications in bioimaging. In this manuscript, we point out a potential problem arising from the application of the object scattering potential in TPM as introduced by Emil Wolf in 1969 for the retrieval of the 3D Refractive Index (RI) distribution of the biosample, a scattering potential that relies on the contemplation of an isotropic RI distribution of the object. Namely, biological samples are bounded by lipid bilayer structures that are well-known to exhibit optical anisotropy dependent on lipid molecular structure, ambient temperature and light wavelength. We have introduced an advanced Mie scattering model for analyzing light scattering off a liposome-lipid membrane assembly and analysis thereof shows that the nanoscale organization of lipids in the observation of cellular endocytosis with polarized light induces a significant change in far-field scattering. We thereby hypothesize that lipid-induced optical anisotropy can significantly impact on interference microscopy studies that only rely on considerations of isotropic 3D RI distributions. Extension of the object scattering potential applied in TPM to include optically anisotropic samples could therefore present a required evolution in TPM.